The invention relates to methods for the determination of the concentration of an analyte in a sample suspected of containing the analyte. More particularly, the invention relates to reducing the effect of interfering substances on measurements conducted during the above methods for the determination of the concentration of an analyte in a sample.
Hydrophobic compounds such as, for example, drugs, vitamins such as, e.g., vitamin D and vitamin B12, and hapten hormones, are often found in the hydrophobic core of lipoproteins, whose surrogate analyte marker is cholesterol, which resides in all lipoproteins including low density lipoprotein (LDL), very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), high density lipoprotein (HDL) and chylomicrons.
As indicated above, vitamin D is one such hydrophobic compound. After formation of vitamin D by UV Light, it is transported by lipoproteins and vitamin D binding proteins (VDBP) in the blood. In assays for vitamin D where vitamin D is not extracted using an organic solvent such as, for example, an alcohol, vitamin D is released from VDBP using a suitable releasing agent. However, vitamin D molecules in the core of lipoproteins are left in the core of the lipoproteins and are untouched for the most part. Extraction assays are laborious and, as mentioned above, involve the use of organic solvents on a sample. Therefore, most immunoassays for vitamin D or other hydrophobic compounds are negatively impacted by one or more interfering substances such as, for example, cholesterol and lipoproteins in a sample because vitamin D molecules that are not released from the interfering substances such as, for example, the core of lipoproteins, are not accessible to an antibody used in an immunoassay. The actual amount of vitamin D in a sample may not be accurately determined and the amount of vitamin D observed in an immunoassay may be falsely elevated or suppressed.
Assessing vitamin D levels in biological samples is important since vitamin D deficiency is related to a number of disorders in mammals. There is a continuing need to develop fast and accurate diagnostic methods to measure a level of a hydrophobic haptenic analyte in a sample taken from a patient. The methods should be fully automatable and be accurate even when conducted on samples having various interfering substances. The assay methods should provide an accurate measurement of the amount of the hydrophobic haptenic analyte in the sample while minimizing inaccuracies resulting from interfering substances present in the sample.